Glycolipids from mammalian cells belong to sphingo-glycolipides comprising a lipid portion, so-called ceramide, composed of sphingosine to which long chain fatty acid is bound by an amide bond, a variety of oligosaccharide chains and a sialic acid. Ganglioside is a general term of sphingo-glycolipides comprising a sialic acid. Recent research has proved that, in general, a sialyl sugar chain of ganglioside, most parts of which are located on the surface of animal cells, is oriented outward from the cell surface so as to play an important role in basic vital phenomenons such as cell discrimination, acceptance and response of information, receptor functions to hormones, viruses, bacteria, cytotoxins and so on, intercellular recognition, and also, differentiation, growth, canceration and immunity of cells.
Within the sugar chains, it is found that hemagglutinin, which comprises virus membrane of influenza virus A, is very effectively bound to a ganglioside comprising a five-sugar chain containing a sialyl(.alpha.2-6)galactose sugar chain (see, Japanese Unexamined Patent Publication No. H6-247995).
It is considered that novel means may be provided in the field of research on influenza virus by synthesizing fluorescent derivatives having a hemagglutinin binding activity similar to said ganglioside and resistance to sialidase on the surface of membrane of influenza virus.
It is found that influenza viruses bind to a sialic acid-containing sugar chain which is included in glycolipids and glycoproteins. Influenza viruses are classified in A-, B- and C-types. Viruses A and B are bound to an N-acetylneuraminic acid- or N-glycolylneuraminic acid-containing sugar chain, and virus C is bound to a 9-O-acetylsialic acid-containing sugar chain, by intervention of hemagglutinin therebetween. In particular, influenza virus A is potently infectious so that a clinical importance of this type of discrimination is widely recognized.
Radioisotope methods, which are used for probe of a phenomenon that bacteria, bacteria-generating toxins and viruses are bound to glycolipids, have problems of (1) health care of researchers, (2) necessity of special facilities or systems, (3) necessity of a person in charge of treatment radioisotope, (4) waste disposal and (5) long half-life of radioisotope. In addition, increase of radioactivity is limited, since a higher specific activity of radioactive probe promotes autoclasis of probe. It is said that improvement of detection limit is difficult based on the principle. In recent years, development of techniques on radiationless isotope have been given importance to so that the fluorescent method is regarded as effective.
Production of fluorescence-labeled glycolipid containing a sialyl sugar chain will be useful, since the sugar chain may be used as a reagent for detection, identification and diagnosis of influenza virus A which is infectious. Specifically, it is considered that novel means may be provided in the field of research of interaction between viruses and cells by synthesizing derivatives into which fluorochrome is introduced at a site of lipid portion which is not related to binding of glycolipid to a receptor, and derivatives which retain a native binding activity of influenza virus A to hemagglutinin, which makes it possible to detect and identify viruses as receptor, and to trace its behavior visually.
From the viewpoint, the inventors have already synthesized GM3 which has fluorescein at a ceramide moiety (see, Japanese Unexamined Patent Publication No. H7-309888). However, it was revealed by examination of fluorescence of the obtained compounds that virus-bound GM3 fluorescein had very weak fluorescence in hydrophobic conditions (conditions to determine fluorescence using a densitometer with the adhesion of GM3 to a microplate), which was insufficient to detect influenza virus.
Furthermore, hemaggultinin of influenza virus tends to recognize a sialyl(.alpha.2-6)galactose bond rather than a sialyl(.alpha.2-3)galactose bond of GM3 (TOSAKENKYU NO SAISENTAN (FUKUDA Minoru Ed.; YOSUISYA), page 141, 1996).
As shown above, it is necessary to inspect a variety of derivatives having a binding activity to sialyl(.alpha.2-6)galactose and fluorochrome which does not depend on detection environment.
It is an object of the invention to provide ganglioside derivatives (I) and (V), which have a binding activity to hemagglutinin on the surface of membrane of influenza virus A and fluorochrome to maintain fluorescent properties in hydrophobic conditions, and synthetic intermediates thereof.
Furthermore, it is another object of the invention to provide a reagent to detect influenza viruses using the derivatives.